Use of a night-vision intensifier for direct visualization by eye of far-red and near-infared fluorescence through an optical microscope

ABSTRACT

Featured are methods for the direct microscopic visualization of samples ( 9 ) using a image intensifier ( 140 ) that intensifies low levels of visible light and converts far-red or near-infra-red light to visible light. Also featured are devices and systems for use in the methods of the invention.

RELATED APPLICATIONS

This application claims the benefit of U.S. Provisional Application Ser.No. 60/386,693, filed Jun. 6, 2002 and U.S. Provisional Application Ser.No. 60/391,520, filed Jun. 25, 2002. The entire contents of each of theabove-identified applications are hereby incorporated by reference.

BACKGROUND OF THE INVENTION

Fluorophores that emit far-red or near-infrared (NIR) light (650-1000nm), such as Cy5 [Mujumdar, et al. (1993) Bioconjug Chem 4:105-11.], arenow widely used in basic biomedical research. This popularity is due totheir strong fluorescence, which can be easily separated from that ofother common fluorophores, and the absence of far-red and NIR autofluorescence in many biological materials [Brelje, et al. (1993) MethodsCell Biol 38:97-181; Murphy (2001) Fundamentals of light microscopy andelectronic imaging, pg 378. John Wiley & Sons, Inc., New York, N.Y.].

The human eye can typically detect light in the 400-700 nm range.Without prolonged dark adaptation or extremely intense illumination,human vision is relatively insensitive to light at wavelengths longerthan 650 nm, and entirely insensitive to light at wavelengths above 700nm [Inoue and Spring (1997) Video Microscopy: The Fundamentals, pg 741.Plenum Press, New York, N.Y.]. Thus, nearly all far-red and NIRfluorescence emissions are invisible to the human eye. Consequently, Cy5and similar fluorophores have a significant shortcoming from theviewpoint of the microscopist as they cannot be directly visualized.

Far-red and NIR fluorescence is usually visualized indirectly throughcharge-coupled device (CCD) or video cameras, often with the furtherassistance of computerized digital imaging equipment. Despite the broadwavelength sensitivity of CCD and video cameras, which can reach intothe infrared spectrum, this indirect approach has several drawbacks. CCDand video cameras typically have limited fields of view compared to thefield of view of the microscope to which they are attached. For example,and using the Nikon E800 epifluorescence microscope and PrincetonInstruments MicroMax digital CCD camera as illustration, the microscopeproduces an intermediate image approximately 25 mm in diameter, or about491 mm². This image can be observed in its entirety through theeyepieces, or observed indirectly using the digital camera. the digitalcamera, however, has an active imaging area of about 60 mm² thus, atleast nine digital frames from the camera are required to match themicroscopist's view through the eyepieces. As such, the indirectexamination of large individual specimens or large numbers of specimensusing a CCD or video camera is impractical and time-consuming.

In addition, CCD and video cameras also may require long exposure orsignal integration times in order to produce acceptable images,regardless of wavelength. Long exposure times, however, make itdifficult or impossible to observe rapid phenomena in living specimens,or visually scan large regions of interest. Furthermore, long exposuretimes and a camera's relatively small imaging area can mean thatspecimens must be exposed to excitation light for extended periods oftime. Prolonged illumination by conventional or laser excitation sourcesis harmful to both living and non-living specimens. For example, lightacross the visible and NIR spectrum is known to disrupt cellular anddevelopmental processes [Brelje, et al. (1993) Methods Cell Biol38:97-181; Daniel (1964) Nature 201:316-317; Hirao and Yanagimachi(1978) J Exp Zool 206:365-9; Hegele-Hartung, et al. (1991) Anat Embryol183:559-71; Potter (1996) Curr Biol 6:1595-8; Hockberger, et al. (1999)Proc Natl Acad Sci USA 96:6255-60; Squirrell, et al. (1999) NatBiotechnol 17:763-7; Konig (2000) J Microsc 200:83-104; Tirlapur, et al.(2001) Exp Cell Res 263:88-97.]. Also a phenomenon referred to asphotobleaching is routinely observed in both fixed and live tissues.

It thus would be desirable to provide a new device, system and methodsfor direct microscopic visualization of a sample using lightintensification techniques. It would be particularly desirable toprovide such a device, system and method that would allow directmicroscopic visualization of light in the far-red light range, thenear-infrared light range and/or the visible light range from a sample.Also, it would be desirable to provide such a device, system and methodthat would convert a light image of a sample including light that is ina non-visible light range (i.e., light not typically visible to thenaked eye), such as the far-red and near-infrared light ranges, into alight image that is visible to the naked eye. It also would beparticularly desirable to provide such a device, system and method thatwould allow direct microscopic visualization of a sample beingilluminated by a light source that is being operated at reduced lightillumination levels particularly light levels that otherwise would notbe observable to the naked eye. Such devices, systems and methods alsowould be easily adaptable for use with conventional flourescencetechniques and microscopic imaging/visualization techniques as well aswith conventional microscopic imaging devices used with fluorescence andbright field microscopy, including conventional microscopes.

SUMMARY OF THE INVENTION

The present invention features a methods for direct microscopicvisualization of a sample such as that done during biomedical research,using light intensification techniques. More particularly such methodsprovide a mechanism for direct microscopic visualization of light in thefar-red light range, the near-infrared light range and/or the visiblelight range. In more particular aspects, such method includes providinga device that converts a light image of the sample, which light imageincludes or is composed of light that is in a non-visible light range(i.e., light not typically visible to the naked eye), such as thefar-red and near-infrared light ranges, into a light image that isvisible to the naked eye. In other aspects, the methodology of thepresent invention includes controlling a light source illuminating thesample so the light output is at a level that reduces/minimizes thepotential for biological damage or the like to the sample beingilluminated and intensifying the light from the sample so as to producea light image that is observable to the naked eye. Such methods alsoadvantageously allow a microscopist or user to directly visualize andobserve the sample in real time. Such methods also advantageously allowthe microscopist to visualize or observe the entire, substantially theentire or a major portion of the intermediate image produced by amicroscopic imaging device used in combination with such methods.Moreover, such visualizing or observing can be accomplished in real timeby the microscopist.

According to an aspect of the present invention, there is featured amethod for microscopic visualization of a sample including intensifyingthe light emanating from the sample and directly observing a light imageprovided by the intensified light, more particularly such lightintensifying and observing occurs in real time. In an embodiment of thepresent invention, the method further includes converting light that isemanating from the sample in non-visible light ranges to light that isin the visible light range and wherein said intensifying and observingincludes intensifying the converted light and observing the light imageprovided by the converted intensified light.

In another embodiment, such a method further includes providing a imageconverter that is configured and arranged so as to intensify the lightreceived at an input end and providing an image at an output end. Inaddition, the method includes locating the image converter in theoptical light path between the sample and the microscopist such that thelight from the sample is received at the image converter input end. Inmore particular embodiments, the image converter is located so that aninput face of a light intensifying device is proximal to theintermediate image plane of the microscopic imaging device (e.g. theintermediate image plane of the microscope).

In further embodiments, the method includes providing a plurality of theimage converters that are located in the optical light path so as toprovide a stereoscopic image. More particularly, the plurality of theimage converter is arranged so as to allow binocular vision. As is knownin the art, binocular or stereoscopic vision preserves depth perceptionand allows the methodology of the present invention to be used incombination with dissection and/or micromanipulation techniques. Incontrast, obtaining stereoscopic vision is extremely difficult toreproduce using conventional indirect visualization approaches ortechniques.

In further embodiments, the image converter is configured and arrangedsuch that light being received at the input end that is in a non-visiblelight range is converted so as to provide a light image that is in thevisible light range at the output end thereof. In more particularembodiment, the image converter is configured and arranged to convertlight in the far-red light range or near-infrared light range into avisible light image at the output end thereof. In additional particularembodiments, the image converter intensifies one of the converted lightor the light in the non-visible light range.

In an exemplary embodiment, the image converter includes a night-visionoptical device that is sensitive to light in the wavelength range ofinterest and converts image is made up of low levels of visible light ornear-infrared light (light in non-visible range) focused on its inputface to images that can be directly visualized that the output facethereof. In further exemplary embodiments, the image converter includesa housing inside which is located the night-vision optical device, whichhousing is configured and arranged to the couple the image converter tothe microscopic imaging device and to minimize external stray light frombeing observed that the input face of the night-vision optical device.

Also featured are a systems and devices embodying and/or for use inimplementing such methodology.

Other aspects, embodiments, features and advantages of the inventionwill be apparent from the following detailed description and claims.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A,B are schematic block diagrams illustrating microscopicvisualtization systems according to the present invention with amonocular eyepiece (1A) and a binocular eyepiece (1B).

FIG. 2A is schematic view generally illustrative of the basic elementsof a microscope.

FIGS. 2B,C are schematic views illustrating placement of a visualconverter according to the present invention at the intermediate imageplane of the microscope (2B) and at a re-positioned intermediate imageplane (2C).

FIG. 3 is a schematic cross-sectional view of a visual converteraccording to the present invention.

FIG. 4A is a cross-sectional view of a shell for a housing according toan embodiment of the present invention.

FIGS. 4B, D are bottom and cross-sectional views respectively and of acap for a housing according to an embodiment of the present invention.

FIG. 4C is a top view of the shell of FIG. 4A.

FIG. 5 depicts the Visualization of Far-Red and Near-infraredFluorescence. Human GFP-EBD cells were stained with the indicatedfluorescent dyes and visualized by epifluorescence on a Nikon TE200inverted microscope through standard eyepieces (By Eye) or through theNIRIC. Cells are also shown by Hoffman Modulation Contrast microscopy(IMC, transmitted light) as seen through standard eyepieces. A 60×, NA0.70 HMC objective was used, final magnification for all images is 150×.HO, Hoechst 33258; GFP, green fluorescent protein; Mito, MitoTracker RedCM-H₂XRos; NIR, near-infrared; LP, longpass; nm, nanometers. Emissionwavelengths imaged for each fluorophore are indicated. The NIRIC detectsblue fluorescence at the lower limit of its spectral range, and producesamplified images in green and red. Far-red and NIR fluorescence is notvisible by eye, but can be clearly seen through the NIRIC. Anessentially complete absence of background fluorescence in NIRIC imageswas noted in the far-red and NIR.

FIGS. 6A-6C depicts the Visual Scanning of Tissue Sections. Using theNIRIC, tissue sections were screened for the presence of DiR-labeledcells on the E800 microscope (epifluorescence, 780 nm longpass) using a10×, NA 0.30 phase objective. Dashed white lines indicate the edges ofthe tissue section. (A) DiR-positive cells were easily seen with theNIRIC (final magnification 10×), and large tissue sections could bequickly surveyed by eye in real time. Box indicates the area shown in B.(b) Grid overlay (red) indicates 25 individual frames captured by aMicroMax CCD camera with a 20×, NA 0.50 phase objective (finalmagnification 20×; the CCD camera failed to detect positive cells usingthe 10× objective, even with long exposure times). Only one frame,outlined in red, shows DiR-positive cells. Most of the CCD frames areempty, and more than 35 frames would be required to image the areaencompassed by the NIRIC at lower magnification. (C, D) The highlightedframe in B, showing DiR-positive cells by epifluorescence (C) and DICmicroscopy (D) as captured with the MicroMax CCD camera (finalmagnification 20×).

FIGS. 7A-7B depict the Visualization of Embryos in Low Light. Mouseembryos were imaged on the TE200 microscope using a 40×, NA 0.60 HoffmanModulation Contrast objective (transmitted light, final magnification100×). (A) With the aid of the NRIC, embryos were visible atillumination levels too low to allow visualization through standardmicroscope eyepieces. (B) At higher illumination levels the embryos werevisible by eye through the microscope's standard eyepieces, asphotographed with the U-III camera (Kodak ELITE Chrome 35 mm slidefilm). A slight degradation of image quality with the NIRIC in thisbrightfield application was observed (compare images at top and bottomarrows).

FIGS. 8A-8C depict epifluorescence microscopy with limited excitationlight using a microscopic array of fluorescent beads immobilized insucrose, which was constructed by micromanipulation. (A) A 20×, NA 0.50objective was used to observe the array on the E800 microscope (finalmagnification for all images is 50×). A portion of the bead standardarray as seen by eye through standard eyepieces (and photographed withthe U-III camera), showing all beads by Nomarksi DifferentialInterference Contrast microscopy (DIC) and their correspondingepifluorescence in green (FITC filters). Relative fluorescenceintensities for the beads in green (FITC filters) and red (TRITCfilters) are indicated. (A, B, C) Bead 8 is unlabeled (nofluorochromes); intensities are relative to the background fluorescenceof Bead 8. Dark arrowheads indicate visible array lines, open arrowheadsmark the location of array lines not visible to observers. (B) Lines ofthe bead standard array visible with FITC filters at decreasing levelsof epifluorescence illumination, through normal microscope eyepieces (ByEye) and the NIRIC. (C) Lines of the bead standard array visible withTRITC filters at decreasing levels of epifluorescence illumination,through normal microscope eyepieces (By Eye) and the NIRIC. (B, C)Illumination from a 100 watt mercury arc lamp was attenuated withneutral density filters. ND, neutral density; ND1, full illumination;ND10, 10% transmission (i.e. 1/10^(th) of the full illuminationintensity); ND100, 1% transmission. Note halo and other artifacting ofbright beads with the NIRIC under strong illumination.

FIGS. 9A-9B depict the direct visualization of Cy5 signals in FISH.Metaphase chromosomes were prepared for fluorescence in situhybridization (FISH). Fluorescent signals were imaged on the E800microscope, final magnification 250×. (9A) Upper panels: Enhancedvisibility of dim FITC signals seen with the NIRIC as compared tostandard eyepieces (By Eye). Lower panels: Chromosomes counterstainedwith DAPI. (9B) Upper panels: Cy5 signals invisible through standardeyepieces (By Eye) are directly visualized with the NIRIC. Arrowheadshighlight chromosomes hybridizing with Cy5-labeled probe. Lower panels:Chromosomes counterstained with DAPI.

DETAILED DESCRIPTION OF THE INVENTION

Referring now to the various figures of the drawing wherein likereference characters refer to like parts, there is shown in FIGS. 1A,Bschematic block diagrams illustrating microscopic visualization systems100 a,b according to the present invention. Reference also is made toFIGS. 2A-C which are schematic views of the basic elements of aconventional microscopic imaging device 10 such as a microscope (FIG.2A) and schematic views illustrating placement of a visual converter 140(also referred to interchangeably herein as an “image converter”)according to the present invention at the intermediate image plane 04 ofthe microscopic imaging device 10 or microscope (FIG. 2B) and at are-positioned intermediate image plane 06 of the microscopic imagingdevice (FIG. 2C). Each of the illustrated microscopes/microscopicimaging devices 10 are positioned so as to view a sample or specimen 8that is located at the specimen plane 9. The specimen is illuminated bya light source 130. The light source 130 is any of a number of lightsources known to those of skill in the art by which a user canilluminate a specimen or sample using a microscope/microscopic imagingdevice.

There is shown in FIG. 1A, a microscopic visualization system 100 a thatincludes a microscopic imaging device 10, a visual converter 140according to the present invention and a light source 130. Themicroscopic imaging device is any of a number of devices or apparatusesknown to those skilled in the art by which a user can directly and/orindirectly observe the specimen including but not limited toconventional microscopes as well as those configured and arranged toallow direct visualization and indirect visualization and/or acquisitionof a permanent record (e.g., picture, digital images, video images).Such microscopic imaging devices also includes those intended for use inboth fluorescence and low light level brightfield microscopy.

The visual converter 140 is operably and optically coupled to themicroscopic imaging device using any of a number of techniques known tothose skilled in the art such that light from the specimen 8 is coupledto the proximal or front end 142 and so that the distal or back end 144of the visual converter is towards the observer. The mechanism foroperably and optically coupling also preferably provides a mechanism tominimize or eliminate the potential for stray external light to enterinto the visual converter 140 via the front end 142 thereof.

In further embodiments, the visual converter back end 144 is configuredand arranged so as to include one or more optical elements 146 a as isknown to those skilled in the art so as to further magnify the imagebeing outputted by the visual converter 140. In a particularillustrative embodiment, the microscopic visualization system includes amonocular eyepiece 150 a as is known to those skilled in the art and thevisual converter back end 144 is configured and arranged so themonocular eyepiece 150 a is operably and optically coupled to the visualconverter back end. The eyepiece 150 a provides a mechanism by which theimage being outputted by the visual converter 140 can be magnified bythe user. Preferably, the visual converter back end 144 is configuredand arranged such that the monocular eyepiece is removable so a user caneasily adjust the amount of magnification.

Referring now also to FIG. 3 is a schematic cross-sectional view of avisual converter according to the present invention, which includes ahousing 150 and a light intensification device 170 or lightintensification tube. The light intensification device 170 is arrangedwithin the housing 150 so that an input face 172 of the imageintensification device is towards the specimen when the visual converter140 is operably coupled to the microscopic imaging device 10 and so anoutput face 174 thereof is arranged towards the observer, viewer oruser.

The light intensification device is any of a number of lightintensification devices known in the art, more particularly devices thatalso are capable of sensing light that lies outside the visible rangeand can generate or provide an image that lies within the visible rangeof the light that is outside the visible range. In more specificembodiments, the light intensification device is any of a number ofconventional night vision tubes or devices. Modern night visionequipment is based on gallium arsenide (GaAs) image intensifier tubes,which convert a wide spectrum of incident light into an amplified streamof electrons that is accelerated onto a phosphor screen, which thenemits visible light. The principles of operation of these types of imageintensifiers are further detailed by Inoue and Spring ((1997) VideoMicroscopy: The Fundamentals, pg 741. Plenum Press, New York, N.Y.). Inan illustrative exemplary embodiment, the light intensification deviceis a FS9925D intensifier tube as manufactured by ITT Industries, havinginput and output faces that are 25 mm in diameter. The intensifier ispowered by 3 volts DC, such as that which can be provided by two AAbatteries, and is sensitive to light from about 450 to 900 nm.

The housing 150 also is configured and arranged so that light that makesup the intermediate image is passed through the front end 142 to thelight intensification device input face 172 and so that the imageprovided at the image intensification device output face is observablefrom the back end 144. In more specific embodiments, and as indicatedherein, the end of the housing 150 corresponding to the visual converterfront end 142 is configured and arranged so as to operably and opticallycouple the visual converter 140 to the microscopic imaging device. Suchcoupling includes mechanical coupling via a screw type connection, aslide fit connection or other any other mechanical connecting techniquesknow to those skilled in the art.

In addition, the housing is constructed so as to secure theintensification device 170 within the housing so as to generallymaintain the input face 172 thereof in fixed relation with respect tothe converter front end 142. In more particular embodiments, the housing150 is constructed and arranged such that the input face 172 is disposedat, about or proximal the intermediate image plane 4 of the microscopicimaging device 10.

In further embodiments, the housing s constructed of materials that areopaque to the light wavelengths or frequencies of use and sized so as tosufficient mechanical strength for the intended use. Such materialsinclude, but are not limited to, metals such as aluminum and plastics.

Now with reference to FIG. 2C, the visual converter 140 further mayinclude one or more optical elements 148 as is known to those skilled inthe art by which the intermediate image plane can be repositioned. Inthis way, a user can adjust the position of the intermediate image planeto optimize it for a given application and for the construction andarrangement of a given visual converter. For example, a particularvisual converter might be constructed in such a way that the input facewould not be proximal to the intermediate image plane of a particularmicroscopic imaging device. Thus, the additional optical components 148can be utilized so as to reposition the intermediate plane so that it isat, about or proximal the input face.

Referring now to FIGS. 4A-D, there is shown a particularly illustrativeembodiment of a housing for a visual converter 140 according to thepresent invention. In the illustrated embodiment, the housing includes ashell 252, a cap 254, and a compression washer (not shown). An imageintensification device 170 is disposed within an interior compartment253 of the shell 252 so image intensification device input face 172 ispositioned so as to face the front end through aperture 254. Inaddition, the housing further includes a ring 256, such as a delrinring, that blocks stray reflected light and forms a seating surface forthe input face 172.

The cap 254 is secured to the back end of the housing shell 252 usingany of a number of techniques known to those skilled in that art. In anillustrative embodiment, the cap 254 is secured to the shell by screws.The compression washer in combination with the cap 254 provides amechanism for securing the image intensification device 170 within theshell interior compartment 253. The cap 254 also is configured andarranged so as to include a through aperture 255 in which is receivedthe monocular eyepiece 150 a. In further embodiments, the cap 254includes a notch 257 therein, through which power leads for the imageintensification device can be routed.

Referring now to FIG. 1B, there is shown a microscopic visualizationsystem 100 b that includes a microscopic imaging device 10, a pluralityof visual converters 140 and a light source 130. Reference shall be madeto the foregoing discussion for FIG. 1A as to the details of themicroscopic imaging device 10, the visual converter 140 and the lightsource 130. In the illustrated embodiment, the microscopic visualizationsystem 100 b is particularly configured for use with a microscopicimaging device configured to provide binocular or stereoscopicvision/images. In this embodiment, a visual converter is disposed ineach of the optical paths operabally and optically coupled to thebinocular eyepieces 150 b, or to two separate eyepieces configured orarranged to provide stereoscopic or binocular viewing.

As is illustrated in either of FIGS. 1A,B the microscopic visualizationsystem 100 a,b can include a microscopic imaging device 10 that isconfigured and arranged so as to have an additional optical port 11 thatis optically coupled so as to also view the specimen 8 at the same timethe specimen is being directly visualized by the user or microscopist.In this way, the microscopist can utilize the one or more other opticalports 11 for indirect visualization of the specimen as well as forobtaining digital, film or video images or at least portions of thespecimen being visualized. For example, a microscopist can directlyvisualize the intermediate image in its entirety, identifying thoseareas of the intermediate image for digital imaging and acquiringdigital images for these specific areas. This should be less costly andless time consuming as compared to conventional indirect imagingtechniques in which large number of digital images are acquired toincrementally image the entire intermediate image.

The methodology of the present invention can be best understood from thefollowing discussion and reference to the systems 100 a,b and visualconverters described herein and shown in FIGS. 1-4. More particularlythe following.

First is described a method for visualization of far-red andnear-infrared (NIR) emitting fluorophores, more particularlyvisualization of far-red and NIR emitting fluorophores by the need tolocalize small numbers of Cy5-labeled cells in large tissue sections.The Cy5 label is chosen to avoid visible spectrum autofluorescence inthe sections. A possible solution, based on the use of sub-optimalexcitation and emission filter sets that focus on the visible portion ofCy5 fluorescence, has been proposed [Ferri, et al. (2000) J HistochemCytochem 48:437-44]. However, this approach cannot be applied if the Cy5signal must be separated from fluorescence or autofluorescence in theorange-red region of the spectrum, and cannot be applied to otherfluorophores with little or no emission in the visible spectrum. Avariety of fluorophores in addition to Cy5 emit light at wavelengthslonger than 700 nm [Haugland (2001) Handbook of Fluorescent Probes andResearch Chemicals, 8^(th) Edition], and have therefore been useless instudies that require visual observation. A general method for the directvisualization by eye of far-red and NIR fluorescence would allow the useof these fluorophores in virtually any fluorescence technique.

The direct visualization methodology of the present invention forvisualization of far-red and NIR fluorescence is advantageous in thefollowing respects: The method is broadly applicable, and not specificto any particular fluorophore, fluorescence technique, or microscope andallows real-time observation and visual scanning, with the largestpossible field of view (FOV). Also, the method is particular suitablefor direct observation or eye viewing, and can be used in combinationwith existing photodocumentation, such as digital imaging using CCDcameras and/or far-red and infrared sensitive photographic photography.Moreover, the method and devices used therewith are easily adaptable foruse with an existing microscope, and within established experimentalprocedures or techniques (i.e., substantive procedural modificationsshould not be required).

As indicated herein, the difficulty in visualizing long-wavelengthfluorescence is due to the physiological limitations of the human eye,not the microscope per se and thus any attempt to make long wavelengthfluorescence directly observable to the human eye must address thisfundamental restriction of human physiology. Accordingly, themethodology of the present invention contemplates converting amicroscope intermediate image composed of invisible long wavelengthlight to an image made up of light in the visible spectrum. A particularmechanism contemplated for use in or for accomplishing this conversionprocess are most the electro-optical devices of the type found in ‘nightvision’ equipment, which has been widely used in military applications.Modern night vision equipment is based on gallium arsenide (GaAs) imageintensifier tubes, which convert a wide spectrum of incident light intoan amplified stream of electrons that is accelerated onto a phosphorscreen, which then emits visible light.

In further embodiments, the method includes bringing the intermediateimage of the microscope/microscopic imaging device 10 to focus on avisual converter 140 containing an image intensifying device 170. Theimage intensifying device 170 typically is highly sensitive to a broadrange of light wavelengths, including but not limited to near-infraredlight, far-red and extremely low levels of visible light which areinvisible to the human eye. The image intensifying device 170 convertsthe invisible intermediate image (image composed of light outside thevisible range) into an image easily visible to the human eye. Theresulting image may be viewed through the visual converter with orwithout additional magnification, such as that provided by a standardmicroscope eyepiece 150 a.

Extremely low levels of visible light (about 400 to about 700 nm) andnear-infrared light (about 700 to about 1200 nm) are invisible to thehuman eye and an observer cannot directly view images made up of thesetypes of light. Thus, direct visual examination of specimens through anoptical microscope is not possible if the specimen is illuminated by oremits low levels of visible light, or near-infrared light. Visualexamination of photosensitive or thermally sensitive specimens, such aslive cells and tissues, or reactive or fluorescent materials, isextremely difficult when the level of light required for direct visualobservations will heat, damage, destroy, or otherwise affect thosespecimens. Visual examination of specimens (or features withinspecimens) that transmit, reflect or emit near-infrared light, such aslong wavelength fluorophores used in many clinical, diagnostic, andexperimental fluorescence techniques, also is not possible since thislight is invisible to the human eye. As further described herein. Themethods of the present invention overcome such shortcomings and allowsuch specimens to be directly visualized by the human eye in real-time,without the need for significant modifications to optical equipment,techniques, or procedures, and without the need for modifications toestablished clinical, diagnostic, or experimental techniques.

As indicated herein, image intensification for the purpose of directvisualization by eye of images made up of low levels of visible light ornear-infrared light has not previously been applied to opticalmicroscopy. Currently, such images are viewed indirectly with the aid ofcharge coupled devices (CCDs) or video cameras; these indirectapproaches have several shortcomings. CCD and video cameras have limitedfields of view when compared to the field of view of the microscope towhich they are attached. This limitation makes the examination of largeindividual specimens or large numbers of specimens through the opticalmicroscope impractical and time-consuming. In addition, CCD and videocameras may require long exposure or signal integration times in orderto produce an acceptable indirectly viewed image. This requirement alsomakes examination of large numbers of specimens difficult orimpractical, and prevents real-time observation through the microscope.As described herein, the methods of the present invention make possiblethe direct, real-time viewing by eye of normally invisible images, usingup to the full field of view of the optical microscope to which it isapplied. When appropriately applied, the method easily and simply allowsbinocular (stereoscopic) vision, which preserves depth perception intechniques such as dissection and micromanipulation; stereoscopic visionis extremely difficult to reproduce by indirect visualizationapproaches.

In particular embodiments, a visualization method according to thepresent invention can be applied to any microscope that produces a realintermediate image. Such a method includes determining the location ofthe intermediate image plane of the microscope to which the method willbe applied and making the intermediate image plane of the microscopephysically accessible. In one embodiment, this can be accomplished byremoval of one or more microscope eyepieces. In further embodiments,such a method further includes bringing the intermediate image intofocus on a visual converter (described elsewhere herein) constructed toallow the visualization by eye of the images or wavelengths of light ofinterest. This step can further included placing the visual converter atthe intermediate image plane (e.g., see FIG. 2B), or moving orrepositioning the intermediate image plane to a conveniently positionedor mounted visual converter. As indicated herein, such re-positioningcan be accomplished by use of additional components 1, including but notlimited to lenses, fiber optics, or image relay systems, depending onthe configuration of the microscope or microscopic imaging device 10 beused in combination with the visual converter 140.

As to the preparation of the specimens to be viewed, examined orobserved, such specimens are prepared normally in all respects usingstandard methods known to those of skill in the art. The modifiedmicroscope is operated normally in all respects, except that theintermediate image may be viewed through the visual converter 140, whichwill allow the direct visualization of images not normally perceptibleby eye. Images rendered visible by the visual converter 140 may beviewed with magnification (such as that provided by the originalmicroscope eyepiece) using additional lenses or components.

The following describes one exemplary use of the methodology and visualconverter 140 of the present invention with a Nikon E800 microscope(described in further detail herein). The image intensifier comprisingthe image intensifying device 170 is an ITT Industries FS9925 seriesintensifier, with input and output faces 25 mm in diameter. Theintensifier is powered by 3 volts DC, provided by two AA batteries, andis sensitive to light from about 450 to 900 nm.

The intermediate image of Nikon E800 microscope microscope is 25 mm indiameter, and the microscope was rendered accessible by removal of oneeyepiece and eyepiece tube. The intermediate image was brought to focuson the input face 172 of the image intensifier by placement of thevisual converter 140 at the intermediate image plane 4 (FIG. 2B). Themicroscope can be operated normally, with the addition that intermediateimages of specimens of interest may be viewed through the visualconverter 140. This application of the method of the present inventionrenders images produced by epifluorescence microscopy utilizing theFluorophores Cy5, DiD, and DiR, which are normally invisible to thenaked eye, easily visible.

The following describes another exemplary use of the methodology andvisual converter 140 of the present invention with an Olympus SZH10stereomicroscope. The intermediate image of the Olympus SZH10stereomicroscope was rendered accessible by removal of one or botheyepieces. The visual converter is constructed with a housing 150dimensioned such that the front end thereof can be directly insertedinto the microscope's eyepiece tube in place of a previously removedeyepiece. The intermediate image was brought to focus on the input face172 of the image intensifier within the visual converter 140 byplacement of an appropriate lens or lenses 148 at the front end of thevisual converter housing 150 as illustrated schematically in FIG. 3. Theconverted intermediate image can be viewed with the aid of magnifyinglenses 146 placed at the back end of the visual converter housing 150.The microscope can be operated normally, with the addition thatintermediate images of specimens of interest may be viewed through thevisual converter 140. In order to preserve stereoscopic vision throughthe microscope, the method is applied to both the left and right opticalpaths, i.e. with the removal of both the left and right eyepieces andthe use of two visual converters such as that illustrated schematicallyin FIG. 1B. Thus, no other steps are required to preserve stereoscopicvision.

The methods of the present invention are adaptable for use with anymicroscope that produces a real intermediate image, including, but notlimited to, focal length, infinity-corrected, compound, epifluorescence,polarization, brightfield, darkfield, phase, interference or modulationcontrast, upright, inverted, and stereomicroscopes with monocular orbinocular eyepieces. The visual converter described may be interchangedfor a standard microscope eyepiece or may be permanently or temporarilymounted to the microscope. The method will result in the conversion ofimages made up of near-infrared or extremely dim visible light into fullfield, real-time images that are easily visible by eye, allowing rapidvisual scanning of a wide variety of specimens.

For the purposes of fluorescence microscopy, the method of the presentinvention is adaptable and contemplated for used in the directvisualization by eye of fluorophores that emit light in thenear-infrared spectrum, such fluorophores include but not limited toCy5, Alexa 647, and DiR. See also the further examples in Table 1 below:TABLE I Emission Fluorphore Maxima (nm) Specificity TOTO-3 660 DNA dyeTO-PRO-5 770 DNA dye LDS 751 712 DNA dye Alexa Fluor 680 707 (Variousconjugates) MitoFluor Far 710 Mitochondrial stain Red 680 FM 4-64 734Lipophilic dye DiD 665 Lipophilic dye DiR 780 Lipophilic dyeCarboxynaph- 668 pH indicator dye thofluorescein RH 237 782 Membranepotential probe(see Haugland, R. P. (2001). Handbook of Fluorescent Probes and ResearchChemicals.)

The method of the present invention also are contemplated for use ininstances where the visualization of near-infrared light is necessary,including but not limited to efforts to avoid background fluorescence orautofluorescence, or to provide spectrally distinct signals in schemesrequiring multiple fluorescence colors, such as Fluorescent In SituHybridization (FISH), Comparative Genomic Hybridization (CGH),microarray hybridization, or multicolor labeling using antibodies ordyes. The method of the present invention also is contemplated for useand adaptable to view extremely dim images made up of light in thevisible spectrum, including but not limited to instances when thefluorescent signal is extremely weak, or when the fluorescence emissionis reduced because the energy of excitation light is or has been limitedin order to avoid damage, heating, or other deleterious effects to thespecimen(s) of interest.

In the case of transmitted or reflected light microscopy, the method ofthe present invention will allow viewing of dim visible spectrum images,including but not limited to those produced by polarized light, orinstances where illumination is weak or has been reduced in order toavoid damage, heating, or other deleterious effects to the specimen(s)of interest. The method of the present invention also is contemplatedfor use in schemes in which illumination is achieved using onlynear-infrared light, such as transmitted or reflected near-infraredmicroscopy.

The method of the present invention also is adptable for use in theultraviolet or infrared (thermal) spectra by a modification of thespectral sensitivity of the image intensifier in the visual converter140 or use of an image intensification device particularly suited forsensing light in the desired range of wavelengths. The method of thepresent invention also is adatable for use with images of varyingbrightness by adjustment of the sensitivity of the image intensifier.The method of the present invention also is adpatable and contemplatedfor use to simultaneously display multiple spectrally distinct colors tothe viewer by the addition of bandpass filters to the visual converter140, in a manner analogous to multiple color fluorescence microscopyusing multiple bandpass filters.

The methods, device and systems of the present invention are furtherillustrated by way of the following examples, which should not beconstrued as limiting. The contents of all references, patents, andpublished patent applications cited throughout this application, as wellas the sequence listing and the figures, are incorporated herein byreference.

EXAMPLES

Materials and Methods

Image Intensifier

A model FS9925D image intensifier tube and power supply were obtainedfrom ITT Industries Night Vision (Roanoke, Va.). The FS9925D is aGeneration 3 gallium arsenide photocathode-based, proximity-focused,non-inverting image intensifier with flat fiber optic input and outputphosphor windows 25 mm in diameter. The intensifier tube has spectralsensitivity from approximately 450 to 900 nm (sensitivity increases withwavelength), and high spatial resolution (the unit used in the presentwork was capable of resolving 64 line pairs/mm, which corresponds to apixel size of approximately 8 μm). Nominal tube specifications are asfollows: Visual quality—no spots larger than 0.003 inch. At 900 volts,the photocathode sensitivity is 2509 mA/lumen (luminous, 2856° K) and147 mA/Watt (radiant, 880 nm). The microchannel plate voltage(40,000×Gain) is 926 volts, and the clamp voltage is 35 volts. Themaximum operating voltages are: photocathode to microchannel plate: 920volts; microchannel plate to output: 1200 volts; and microchannel plateoutput to screen: 4200 volts.

Three (3) volts DC current was supplied to the intensifier power supplyfrom two ‘C’ batteries in series, operated by a simple on/off switch.Wire leads to the intensifier were extended at the factory byapproximately one meter to allow convenient remote placement of theswitch, batteries, and power supply. The intensifier gain was alsofactory preset to 40,000 fold. In operation, the intensifier convertsincident light within its operating spectral range at its input faceinto green light emitted at its output phosphor face without spatialdistortion.

Hardware

A housing comprised of a two-part shell (shell proper and cap) wasconstructed to house the intensifier tube. The shell protects theintensifier tube, blocks stray light, allows a magnifying eyepiece to beplaced at the intensifier output window, and facilitates the coupling ofthe intensifier to a microscope. The shell is open at the bottom toallow incident light to reach the intensifier input optic and at the capto allow viewing of the output phosphor. The intensifier tube fitsinside the shell such that its input optic faces the bottom opening, andis held in place by the cap with the aid of a compression washer. Theintensifier output phosphor is visible through the opening in the cap.The cap was also slotted to allow passage of the intensifier powerleads.

The body of an Olympus WH1 Ox-H/22 focusing eyepiece was shortened suchthat when the eyepiece is appropriately placed at the shell cap theintensifier output window lies at the eyepiece reticle landing (i.e., atthe normal location of the microscope intermediate image). Forflexibility in coupling to various microscopes, the opening at thebottom of the shell was dimensioned and threaded as a standard femaleC-mount, but was not used in this manner for the present work. Theassembled prototype—shell, intensifier tube, cap, and eyepiece—isreferred to alternately herein as the “near-infrared image converter”,or “NIRIC”. It should be noted that the general term “image converter”is interchangeable herein wit the term “visual converter”.

Due to its size, the NIRIC could not be practically mounted on amicroscope in the most logical manner as a modular replacement eyepiece.However, the NIRIC could be used as a ‘third eyepiece’ when placed atthe camera port of a microscope's trinocular head. In thisconfiguration, all three eyepieces provide equivalent views to anobserver: The microscope intermediate image can be viewed at 10×magnification through the microscope's standard eyepieces, while theNIRIC conducts the intermediate image focused onto its input windowwithout magnification to the output window, which is also viewed througha 10× eyepiece. A parfocalizable coupler that fit the camera ports ofthe two Nikon microscopes used here (see below) was made for the NIRIC,and used throughout the work described.

Microscopes

Two infinity-corrected Nikon Eclipse epifluorescence microscopes (CFI60optics) served as a platform for the NIRIC—an E800 (upright) and a TE200(inverted). Both microscopes use 100-watt mercury arc lamps (UshioUSH-102D) as fluorescence excitation sources, and 12-volt, 100-watttungsten bulbs as illumination for brightfield and transmitted lighttechniques. Both microscopes were equipped with trinocular image heads(eyepieces and one camera port), and beamsplitters to direct images tosecondary camera ports. The E800 was equipped for phase and Nomarskidifferential interference contrast (DIC) microscopy. The TE200 wasequipped for Hoffman Modulation Contrast (HMC) microscopy usingextra-long working distance (ELWD) objectives. During testing, nomodifications were made to either microscope, and no efforts were madeto replace objectives in order to improve light throughput to the NIRIC,i.e., objectives incorporating phase rings or modulator plates were notreplaced with objectives without such modifications. Further details ofthe E800 and TE200 microscopes are provided below in Table 2: TABLE 2E800 TE200 General Upright, infinity- Inverted, infinity- corrected,CFI60 corrected, optics, phase, DIC, CFI60 optics, HMC, epifluorescence;epifluorescence; trinocular trinocular erect image head image headCamera Ports Front and Rear Front and Side Beamsplitters Front 100%eyepieces Front 100% eyepieces 80%/20% 80%/20% camera/eyepiecescamera/eyepieces 100% camera 100% camera Rear: 100% front Side: 100%front beamsplitter beamsplitter 100% camera 80%/20% camera/frontbeamsplitter Eyepieces CFIUW 10×/25 CFIW 10×/22 Condenser NA 0.90 dry NA0.60, HMC ELWD G3, 40 mm working distance Objectives 10×/.30 (PF, 4×/.13(PF,PhL) Magnification/NA Ph1, DIC M) 10×/.30 (PF, HMC, ELWD) 20×/.50(PF, 20×/.45 (PF, HMC, ELWD, Ph1, DIC M) corr) 100×/1.4 (oil, 40×/.60(PF, HMC, ELWD, PA, DIC H) corr) 60×/.70 (PF, HMC, ELWD, corr)Transmitted 12 Volt, 100 Watt 12 Volt, 100 Watt Light Source tungstenbulb tungsten bulb Fluorescence 100 Watt mercury 100 Watt mercury arclamp Excitation Source arc lamp (Ushio (Ushio USH-102D) USH-102D) NotesFive fluorescence Four fluorescence filter filter cubes, cubes neutraldensity filters to attenuate fluorescence sourcePF, PlanFluor,PA, PlanApo;NA, numerical aperture;Ph, phase;DIC, Nomarski Differential Interference Contrast;HMC, Hoffman Modulation Contrast;ELWD, extra-long working distance;corr, correction collar.Optical Filters

Fluorescence filter sets were obtained from Nikon (via Inage Systems) orChroma Technology (Brattleboro, Vt.). The E800 and TE200 microscopes usethe same epifluorescence filter cubes. Filters covering emissionwavelengths from blue to far-red (DAPI, GFP, TRITC, Cy5) were generallyavailable standard sets known to those skilled in the art. For NIRfluorescence emissions we used a custom set designated 780DCXR, made byChroma (excitation filter 667-742 nm, dichroic mirror 780 nm, emissionfilter 780 nm long pass). Neutral density (ND) filters were obtainedfrom Chroma; ND values are referred to herein using Nikon'snomenclature, as follows: ND1=100% transmission, ND10=10% transmission(i.e. 1/10^(th) of the full illumination intensity), ND20=5%transmission ( 1/20^(th)), and so on. Large ND values were obtained bycombining filters in series, with the aggregate values shown. Forexample, ND10+ND100 would be listed as ND1000 ( 1/1000^(th), 0.1%transmission). Further details of the filters used are presented belowin Table 3: TABLE 3 Filter Set Color Excitation DM EmissionFluorochromes DAPI Blue 330-380 400 435-485 DAPI, HO UV-2E/C Blue340-380 400 435-485 DAPI, HO B-2E/C Green 465-495 505 515-555 FITC(FITC) EN GFP Green 450-490 495 500-550 GFP G-2E/C Red 528-553 565600-660 Mito (TRITC) HYQ Cy5 FR 590-650 660 663-735 Cy5, DiD 780DCXR NIR667-742 780 780LP DiRExcitation, DM, and Emission values are in nanometers (nm).DM, dichroic mirror;FR, far-red;NIR, near infrared;LP, long pass.All filter sets except 780DCXR were manufactured by Nikon. 780DCXR is acustom filter set manufactured by Chroma Technology.DAPI and UV-2E/C sets were used interchangeably.Photography, Photomicrography, Cameras, Image Processing

Comparisons of images visible by eye through standard microscopeeyepieces and images visible with the aid of the NIRIC were made bycomparing direct photomicrographs with photographs of the outputphosphor face of the image intensifier. Film photography was used as astand-in for human vision because film's response is specificallytailored to visible wavelengths of light. Differences between film andhuman vision in terms of sensitivity and dynamic range can be partiallycompensated for by controlling exposure time during photography. Thesemeasures are not quantitative, and photographs were chosen to conveywhat was visible when specimens were observed by eye, as judged byseveral individuals. Kodak ELITE Chrome 400 color slide film was usedfor all 35 mm film photography; its spectral sensitivity is essentiallylimited to the range between 400 and 700 nm [Kodak Publication No. E-149(1998)]. Various Nikon SLR camera bodies and lenses were used for studioand lab photography. A MicroNikkor 60 mm macro lens was used tophotograph the NIRIC's output phosphor face. Photomicrographs from boththe E800 and TE200 microscopes were taken with a Nikon U-III automaticphoto system.

Photodocumentation of images from the NIRIC's output phosphor wasperformed using a 35 mm single lens reflex (SLR) camera (with a lock-upmirror) and a 60 mm macro lens, using a tripod. The eyepiece and cap ofthe NIRIC's shell were removed, and the SLR aimed at the NIRIC outputphosphor from directly above. Pictures were taken in a totally darkenedroom. The macro lens was focused so as to fill the film frame (nominally24×36 mm) with the image of the NIRIC's phosphor face (25 mm diameter);the resulting images were essentially actual size.

Direct photomicrography was performed with a Nikon U-III 35 mm automaticphoto system. In all cases, microscope beamsplitters were used to sendequal amounts of light to the NIRIC and the U-III. Photographs from theNIRIC and the U-III were taken simultaneously, with the NIRIC at the‘third eyepiece’ position and the U-III at the secondary camera port.Microscope intermediate image magnifications to both devices wereexactly matched with the use of identical Nikon PLI 2.5× projectionlenses. To accommodate the focal distance of the projection lens, acustom phototube was made for the NIRIC. The assembly (projection lens,phototube, coupler, NIRIC) was parfocal with both the U-III system(projection lens and phototube) and the microscope eyepieces. KodakELITE Chrome 400 color slide film was also used for all photography withthe U-III.

Except in cases where fields of view (FOV) were explicitly compared,photographs were cropped to the FOV of the NIRIC. FOV measurements weremade with a calibrated stage micrometer. Developed film was digitized byscanning with a Nikon LS-2000 film scanner at 12 bits per color channel(RGB) and an optical resolution of 2700 pixels per inch. Images werestored in the lossless Tagged Image File Format (TIFF, 16 bit files).Digital images were captured with a Princeton Instruments MicroMax CCDcamera (model RTE/CCD-1300-Y/HS, Roper Scientific, Trenton, N.J.) usingMetaMorph imaging software (Universal Imaging, Downingtown, Pa.).

Fluorescent Stains and Dyes

Hoechst 33258 (HO), MitoTracker Red CM-H₂XRos (Mito), Vybrant DiD (DiD),and DiR were all obtained from Molecular Probes (Eugene, Oreg.). Withthe exception of DiR, these were used essentially as recommended by themanufacturer. DiR was dissolved in pure anhydrous ethanol to 1 mg/mL,and then used according to the guidelines for Vybrant DiD.

In Vitro Live Cell Staining

The production and culture of human embryoid body-derived (EBD) cellsexpressing green fluorescent protein (GFP-EBD cells) has been described[Shamblott, et al. (2001) Proc Natl Acad Sci USA 98:113-8.]. To stainGFP-EBD cells for photography, HO, Mito, DiD, and DiR weresimultaneously added to fresh, prewarmed EGM2MV media (Clonetics, SanDiego, Calif.) and mixed to make staining medium. Normal culture mediumwas removed and replaced by this staining medium; cells were thenreturned to the incubator and stained for two hours. Cells were thenrinsed five times with prewarmed Dulbecco's phosphate buffered saline(DPBS, Life Technologies, Rockville, Md.) to remove excess stain. Thefinal wash was removed and replaced with prewarmed DMEM without phenolred (Life Technologies, Carlsbad, Calif.) or with prewarmed DPBS, bothcontaining 5% fetal calf serum (FCS), 20 mM lactate, and 20 μL/mlOxyFluor (Oxyrase, Inc., Mansfield, Ohio) to reduce photobleaching. Thetissue culture dish was sealed to prevent gas exchange, and cells werereturned to the incubator for 30 minutes. The dish was then removed fromthe incubator and photographed on the TE200 inverted microscope.

Injection of Cells into Mouse Spleen, Cryosectioning

Human EBD cells [Shamblott, et al. (2001) Proc Natl Acad Sci USA98:113-8] were labeled with DiR, trypsinized, and washed three times inDPBS without Ca²⁺ or Mg²⁺ (Life Technologies) to remove unincorporateddye. 1×10⁵ labeled cells in 100 μL DPBS were injected into cadavericmouse spleens through a 27G butterfly needle. Spleens were then fixed in4% paraformaldehyde (Electron Microscopy Sciences, Fort Washington, Pa.)in DPBS at 4° C. overnight. After fixation, spleens were transferredinto 30% sucrose and incubated overnight at 4° C., and then placed inO.C.T. compound (Tissue-Tek, Sakura, Torrance, Calif.) and frozen (at−80° C. in 2-methylbutane). Eight micron sections were cut on a cryostat(Microm, Richard-Allan Scientific, Kalamazoo, Mich.) and transferred toglass slides. Slides were stored at −20° C. until examined.

Fluorescent In Situ Hybridization (FISH)

WAV-17 cells have been described [Patterson (1998); Slate and Ruddle(1978) Cytogenet Cell Genet 22:270-4; Raziuddin, et al. (1984) Proc NatlAcad Sci USA 81:5504-8.], and were grown in DMEM containing 15% FCS,0.10 non-essential amino acids, 100 units/mL penicillin and 100 mg/mLstreptomycin (Life Technologies), at 37° C., 90% humidity, in a 5% CO2atmosphere. Metaphase spreads were made as follows: Rapidly growingWAV-17 cells were incubated in colcemid (0.1 mg/mL, added to culturemedia, KaryoMax, Life Technologies) for 20 minutes, and cells arrestedin metaphase were dislodged by sharply striking the culture dish.Arrested cells were collected, pelleted, and resuspended in 75 mM KCland incubated at 37° C. for 35 minutes. The cells were then fixed in 3:1methanol/acetic acid. Fixed cells were dropped onto glass slides in acontrolled humidity chamber, and allowed to dry. Slides were stored at−20° C. until used for FISH.

To synthesize probes for FISH, human placental DNA (Sigma, St. Louis,Mo.) was biotinylated using a BioNick kit (Life Technologies) accordingto the manufacturer's instructions. Probes were stored at −20° C. untilused for FISH. FISH was carried out as follows: WAV-17 metaphase spreadslides were aged in 2×SSC, dehydrated in ethanol, and air-dried. 100 ngof biotinylated human placental DNA probe in 12 mL of 50%formamide/2×SSC per slide was sealed under a coverslip, and the slideand probe denatured (75° C., 6 minutes) and then hybridized (37° C.,overnight). Slides were washed to a final stringency of 2×SSC at roomtemperature. Hybridized probe was detected with fluorescein-avidin DN(Vector Labs, Burlingame, Calif.) or Fluorolink Cy5-labeled streptavidin(Amersham-Pharmacia Biotech, Piscataway, N.J.) and biotinylatedanti-avidin D antibody (Vector Labs) in 4×SSC containing 0.05% Tween-20and 5% non-fat dry milk as blocking agent. Chromosomes werecounterstained with DAPI Molecular Probes), and the slides mounted withProlong antifade reagent (Molecular Probes). Slides were examined on theE800 microscope with a 100× objective using Cargille Type FF immersionoil.

Embryos

Mouse embryos at various stages of development were maintained indroplets of M16 medium [Hogan, et al. (1994) Manipulating the MouseEmbryo: A Laboratory Manual, 2^(nd) edition, Cold Spring HarborLaboratory Press, Plainview, N.Y.] under oil at 37° C. in a 5% CO₂atmosphere.

Fluorescence Intensity Standards

SPHERO Rainbow Calibration Particles were obtained from Spherotech, Inc.(Libertyville, Ill.). The set (RCP-30-5A-1-8, highly concentratedstocks) is intended for use in flow cytometers, and consists of 3 μmpolystyrene beads of eight different fluorescence intensities. The beadsare calibrated in units of molecules of equivalent fluorophore (MEF)[Gaigalas (2001) Journal of Research of the National Institute ofStandards and Technology 106:381-389.], covering four orders ofmagnitude in intensity.

An ordered intensity array was constructed by micromanipulation asfollows: 50 μL of beads were pelleted by a short spin in a microfuge,washed once in 50 μL of water, and pelleted again. The supernatant wasremoved, and the beads resuspended in 2-5 μL of 30% sucrose (w/v inwater). This wash procedure was carried out separately for each of theeight kinds of beads in the set. Standard #1 glass coverslips werewashed once in isopropanol, once in 100% ethanol, and once m water, andthen aspirated dry. A washed coverslip was placed on a glass slide heldon the stage of the TE200 inverted microscope, which was equipped with amicromanipulator (Narishige, Tokyo, Japan) that controlled a glassmicropipette (12-15 μm inner diameter). A drop (1 μL) of beads in 30%sucrose was deposited near the edge of the coverslip, and the tip of themicropipette lowered into the drop, allowing the bead/sucrose suspensionto front-fill the micropipette by capillary action.

The tip of the micropipette was adjusted to be in contact with thesurface of the coverslip, and the filled micropipette was then used as apen to write a line of beads in sucrose across the coverslip by linearmovement of the microscope stage. This was repeated for all eight kindsof beads, resulting in a closely spaced linear array that progressedfrom the brightest to the dimmest beads. The coverslip was allowed toair dry overnight in the dark, immobilizing the beads in sucrose. Thecoverslip was then mounted on a glass slide, bead side down, using adrop of Cargille Type FF immersion oil (Cargille, Cedar Grove, N.J.) asmounting medium. The edges of the coverslip were left unsealed, and thearrays stored at room temperature in the dark. The sucrose lines of thearray were stable for approximately one week, after which neighboringlines began to intermix.

EXAMPLE 1 NIRIC Construction, Background, and Field of View

The NIRIC and coupler was disposed in the ‘third eyepiece’ position onthe E800 microscope. The output phosphor of the NIRIC is a monochromegreen; therefore the output image is always green regardless of thespectral content of the intermediate image at the intensifier's inputoptic. When the NIRIC is switched on there is a 1-2 second delay beforethe intensifier's output phosphor becomes active. When switched on butnot exposed to light, the NIRIC displays sparse, pinpoint, rapidlychanging random signals very similar to the noise (‘snow’) typicallyseen with CCD and video cameras. The noise level increases slightly asthe intensifier tube heats up with use, but is generally so dim that itis negligible. Measurements with a stage micrometer showed that thefield of view provided by the NIRIC in ‘third eyepiece’ configurationwas identical to the view through the microscope's normal eyepieces forboth the E800 and TE200 microscopes.

EXAMPLE 2 Spectral Performance and Visualization of Far-Red and NIRFluorescence

The NIRIC was tested across its operational range (450-900 nm) byimaging live cells stained with a series of fluorescent dyes. Humanembryoid body-derived cells constitutively expressing high levels ofgreen fluorescent protein (GFP-EBD cells), were simultaneously stainedwith Hoechst 33258 (HO, a membrane-permeant DNA dye), MitoTracker RedCM-H2XRos (Mito, stains active mitochondria), and the lipophilic dyesDiD and DiR. DiD has spectral characteristics very similar to those ofCy5; DiR fluorescence lies in the NIR and has no visible component.Quintuply-labeled cells were imaged with an ELWD 60×, NA 0.70 HMCobjective on the TE200 inverted microscope, with full illumination froma 100-watt mercury arc lamp as the excitation source. Results are shownin FIG. 5.

The blue fluorescence from HO-stained nuclei was so strong that it wasdetected by the NIRIC even though the vast majority of HO fluorescenceemissions are below the operating range of the NIRIC intensifier tube.Although blue signals are detected, the NIRIC does not appear to amplifythem. In green (GFP), and to a greater extent in red (Mito), signalamplification through the NIRIC was apparent. Amplification wasaccompanied by a noticeable loss of resolution in the observed image,consistent with saturation of the intensifier tube. In the far-red(DiD), very little was visible by eye through the microscope's standardeyepieces, but strong, specific cell staining was clearly seen using theNIRIC. In the NIR (DiR), the field was completely dark when observedthrough the microscope's standard eyepieces, but cells stained with DiRwere easily visible through the NIRIC. Sharp punctate staining wastypical for cells stained with DiD and DiR; these dyes appear to berapidly internalized from the plasma membrane and concentrated inendosomes or lysosomes. We noted an almost total absence of backgroundfluorescence in NIRIC images in the far-red and NIR compared tobackground levels at shorter wavelengths. These results demonstrate thatthe NIRIC allows the direct visualization of fluorescence emissions atwavelengths normally undetectable with the naked eye.

EXAMPLE 3 Visual Scanning of Tissue Sections

The NRIC was used to locate small numbers of fluorescently labeled cellsin tissue sections. FIGS. 6A-6D demonstrate the successful use of theNIRIC in streamlining this screening process. DiR-labeled human EBDcells were injected into mouse spleens. The spleens were then processedfor cryosectioning, and examined under epifluorescence on the E800microscope.

With the NIRIC in ‘third eyepiece’ position, sections were scanned atlow power using a 10×, NA 0.30 phase objective. Several widely separatedclusters of bright DiR-labeled cells were clearly visible through theNIRIC in the section shown in FIG. 6A. Attempts to image these cellswith the MicroMax CCD camera were not successful, even with longexposures. In order to compensate for the CCD camera's low sensitivityat DiR emission wavelengths, the section was indirectly scanned using a20×, NA 0.50 objective. Under these conditions, DiR-labeled cells wereimaged by the CCD camera, but more than 35 digital frames would berequired to survey the field visible at 10× through the NIRIC. FIG. 6Bshows the field presented by the NIRIC at 10×, overlaid by a gridshowing some of the individual frames captured by the CCD camera at 20×.All of the empty frames in the grid can be avoided with the aid of theNIRIC, since large areas can be scanned in real time, necessitating onlya few long exposure images from the CCD camera for documentation.

EXAMPLE 4 Visualization of Embryos with Minimal Illumination

The photosensitivity of embryos has been noted for some time [Daniel(1964) Nature 201:316-317; Hirao and Yanagimachi (1978) J Exp Zool206:365-9; Hegele-Hartung et al. (1991) Anat Embryol 183:559-71;Squirrell, et al. (1999) Nat Biotechnol 17:763-7.], and has significantimplications for many areas of basic and clinical research, includingstudies of development and differentiation, transgenesis, and in vitrofertilization (IVF). To limit phototoxicity, investigators typicallyilluminate embryos with longer wavelengths of light. This may entailobservation of embryos and cells under red light [Daniel (1964) Nature201:316-317; Hirao and Yanagimachi (1978) J Exp Zool 206:365-9;Squirrell, et al. (1999) Nat Biotechnol 17:763-7.], or the applicationof multi-photon fluorescence techniques [Squirrell, et al. (1999) NatBiotechnol 17:763-7.]. However, multi-photon techniques require highexcitation energies that are potentially damaging, and there is evidencethat even long wavelength light is harmful to living tissues [Potter(1996) Curr Biol 6:1595-8; Konig (2000) J Microsc 200:83-104; Tirlapur,et al. (2001) Exp Cell Res 263:88-97.].

The NIRIC incorporates an image intensifier 170, and thereby provides asimple and direct way to reduce phototoxicity in sensitive specimens byallowing observation with greatly reduced illumination intensities inboth transmitted light and fluorescence techniques. To qualitativelyassess the NIRIC's performance in low-light brightfield applications weobserved mouse embryos on the TE200 microscope by HMC microscopy(transmitted light) using a 40×, NA 0.60 objective. Illuminationintensity was controlled by reducing the operating voltage of themicroscope's tungsten bulb.

With the NIRIC, embryos could be seen clearly at illumination levelsthat were insufficient for visualization by eye through standardeyepieces. The NIRIC conveyed focusing and stage movements in real timeand without artifacting. FIGS. 7A-7B shows the embryos as seen throughthe NIRIC at low light levels (FIG. 7A), and as seen by eye at higherillumination levels (FIG. 7B, as photographed with the U-III camera).There was a slight degradation of image quality when the embryos wereviewed through the NIRIC (compare images at red and white arrows).However, we found the clarity and resolution of the NIRIC imagesufficient for the vast majority of applications, includingmicromanipulation.

EXAMPLE 5 Epifluorescence Microscopy with Drastically Reduced ExcitationLight

To gauge the sensitivity of the NIRIC in epifluorescence applications,we adapted FACS intensity standards for use with the E800epifluorescence microscope. A linear array of fluorescently labeledbeads was made as described in Materials and Methods. The beads'fluorescence intensities are calibrated in units of Molecules ofEquivalent Fluorophore (MEF) [Gaigalas (2001) Journal of Research of theNational Institute of Standards and Technology 106:381-389.], however wedid not attempt to use these values in our measurements. Instead ofmeasuring the actual intensity of the standard, we assessed the minimumamount of excitation light required to visualize the standards. Thisapproach is relevant in the following way: the most effective way toreduce photobleaching or phototoxicity during fluorescence observationsis to limit exposure to excitation light, either by minimizing the timethe specimen is illuminated or by reducing the intensity of theexcitation light. These measures have disadvantages—limitingillumination time precludes extended observations, and limitingillumination intensity can obscure subtle or dim fluorescent signals.Visualization through the NRIC allows reduction of illuminationintensity without these associated disadvantages. Our measurementindicates how little excitation light is sufficient to illuminate aspecimen observed through the NIRIC.

All sensitivity measurements were carried out on the E800 microscope. A20×, NA 0.50 objective was used to include all eight array lines in thesame field of view. The array was observed through TRITC fluorescencefilters. Excitation light from a 100-watt mercury arc lamp wasconsidered full illumination, and all comparisons refer to thisillumination level. Excitation light was attenuated with a series ofneutral density filters placed between the mercury source and thefluorescence filters. Large ND values were obtained by combiningfilters. Three observers judged which beads were visible at eachillumination level.

The beads and their immobilizing sucrose lines were all visible by DICbrightfield microscopy (FIG. 8A, top). The beads are numbered 1 through8 (brightest to dimmest, respectively), and their relative brightness isindicated. For reference, calibrated MEF values (for equivalentphycoerythrin) are also listed. Beads in group 8 were unlabeled(nonfluorescent), and represent background controls. The group 8controls and the sucrose lines were not visible during epifluorescenceobservations. For reference, calibrated MEF values are listed below inTable 4. TABLE 4 Relative Fluorescence Intensity Bead Green Red MEFLMEPE 1 3300 4286 330,000 300,000 2 1400 1771 140,000 124,000 3 400 48640,000 34,000 4 150 171 15,000 12,000 5 47 54 4700 3800 6 18 17 18001200 7 6 5.7 600 400 8* 1 1 100 70Bead 8 is unlabeled (no fluorochromes);Intensities are relative to the background fluorescence of Bead 8, whenviewed in green (FITC filters) and red (TRITC filters).Calibrated intensity values for FACS:MEFL, molecules of equivalent fluorescein;MEPE, molecules of equivalent phycoerythrin.

FIG. 8B shows results obtained with decreasing illumination intensityfor green emissions (FITC filters), and FIG. 8C shows results for redemissions (TRITC filters). Under full epifluorescence illumination, allseven labeled beads were visible by eye and through the NIRIC (ND1).Pronounced halo and other artifacting of bright beads were produced bythe NIRIC under strong illumination. With a one hundred-fold reductionof excitation light, only the four brightest beads remained visible byeye, while all seven labeled beads were still visible through the NIRIC(ND100). Table 5 summarizes the results at greater attenuation levels.At ND10000 none of the beads were visible by eye, while the NIRICrendered the four brightest beads visible at ND40000, and the twobrightest beads visible at ND100000. These results clearly demonstratethat the NIRIC can be used to facilitate prolonged fluorescenceobservations and minimize photobleaching and phototoxicity bydrastically reducing the amount of excitation light required tovisualize specimens. TABLE 5 Beads visible in Green Beads visible in Red(corresponding to FIG. 8B) (Corresponding to FIG. 8C) ND By Eye NIRIC ByEye NIRIC 1 1, 2, 3, 4, 1, 2, 3, 4, 1, 2, 3, 4, 1, 2, 3, 4, 5, 6, 7 5,6, 7 5, 6, 7 5, 6, 7 10 1, 2, 3, 4, 5 1, 2, 3, 4, 5, 6 1, 2, 3, 4, 1, 2,3, 4, 5, 6, 7 5, 6, 7 100 1, 2, 3, 4 1, 2, 3, 4, 5 1, 2, 3, 4 1, 2, 3,4, 5, 6, 7 1000 1, 2 1, 2, 3, 4 1, 2 1, 2, 3, 4, 5, 6 10000 1 1, 2 — 1,2, 3, 4 20000 — 1 — 1, 2, 3, 4 40000 — 1 — 1, 2, 3, 4 80000 — — — 1, 2,3 100000 — — — 1, 2The table indicates which beads in the Relative Fluorescence IntensityStandard Array are visible through standard microscope eyepieces (ByEye) or with the NIRIC at decreasing levels of epifluorescenceillumination (i.e., increasing attenuation by neutral density filters).Green = FITC filters,Red = TRITC filters;ND = neutral density.ND1, full illumination (no attenuation, 100 watt mercury arc lampsource);ND10, 10% transmission (i.e., 1/10^(th) of the full illuminationintensity);ND100, 1% transmission, and so on.

EXAMPLE 6 Visualization of Far-Red Fluorescence in Fluorescent In SituHybridization

Cy 5 and other long wavelength-emitting fluorochromes are widelyemployed as labels in multicolor fluorescent in situ hybridization(FISH) experiments because their emissions can be easily separated fromthe emissions of visible spectrum fluorochromes. However, their usemandates indirect visualization of hybridization signals, usually with aCCD camera As mentioned previously, this process is often tedious andtime-consuming.

The NIRIC was used to directly visualize fluorescent signals from aCy5-labeled FISH probe hybridized to metaphase chromosomes. WAV-17 cells[Patterson (1998); Slate and Ruddle (1978) Cytogenet Cell Genet22:270-4; Raziuddin, et al. (1984) Proc Natl Acad Sci USA 81:5504-8.]are hyperdiploid mouse-human hybrids that carry an average of threecopies of human chromosome 21 as the only human genetic material (cellsmay carry as few as two or as many as five copies). Total human genomicDNA was biotinylated and used as a FISH probe against WAV-17 metaphasechromosomes. Probe hybridization to human chromosome 21 was detectedwith FITC-labeled avidin or Cy5-labeled streptavidin. Metaphasechromosomes were then examined on the E800 microscope using a 100× oilimmersion objective. The NIRIC enhanced the visibility of dim FITCsignals (FIG. 9A), and allowed the direct visualization of Cy5 signalsthat cannot be seen through standard eyepieces (FIG. 9B). Visualizationwith the NIRIC was accomplished in real-time, and large numbers ofmetaphase spreads were quickly scanned and examined. It should be notedthat the NIRIC cannot compensate for poor contrast (or poorsignal-to-noise ratio) in specimens. Signals from backgroundfluorescence and nonspecific staining of mouse chromosomes areintensified along with specific hybridization signals, and cannot besubtracted from the output image as is possible with CCD cameras.

Our results demonstrate that use of electro-optical image intensifyingdevices such as night vision image intensification devices can bepractically and effectively applied to optical microscopy. The NIRICtested successfully met all design goals: its use required nomodifications to the test microscopes, allowed real-time observation andvisual scanning, and did not restrict the observed field of view. TheNIRIC can be used to visualize any transmitted light or fluorescenceemission that falls within its operating range (450-900 nm). Byconverting far-red and NIR light into green light, the NIRIC allows thedirect visualization of long wavelength-emitting fluorophores whosesignals are normally invisible to the naked eye. Due to its highsensitivity, the NIRIC makes visualization possible under drasticallyreduced illumination levels in both transmitted light andepifluorescence techniques, thereby reducing photobleaching andphototoxicity. Because the NIRIC allows visualization in real time, itcan be used to observe rapid phenomena and motion (e.g., duringmicromanipulation), and to quickly scan various specimens.

It should be recognized that the results reported herein significantlyunderestimate the NIRIC's performance. During the testing of the NIRIC'ssensitivity, the dimensions of the fluorescent bead array required theuse of an objective with limited light-gathering ability (20×, NA 0.50).Tests with higher power, higher NA objectives showed that the NIRICcould operate with far less illumination than indicated by Table 1.Furthermore, the objectives used during testing contained phase rings orwere modified for HMC microscopy, reducing the amount of light conductedto the NIRIC. Lastly, the figures of tthe images of the NIRIC's outputphosphor screen were recorded without the benefit of secondarymagnification provided by the 10× eyepiece, and therefore show afraction of the detail as would have been seen by an observer. Theimages shown here are also less resolved than those seen by an observerlooking through the NIRIC's eyepiece because they have been focusedtwice—once by the microscope onto the intensifier tube's input face, anda second time by the macro lens of the SLR camera used fordocumentation.

Although the shell and other coupling hardware described here fit theNIRIC onto Nikon infinity-corrected microscopes, the NIRCI and itsdesign principles can be extended to any microscope whose intermediateimage can be focused onto the input optic of an image intensifier tube.Even techniques requiring stereoscopy can be accommodated, if twodevices can be arranged in a binocular configuration. The NIRIC can alsobe modified for use with blue-emitting fluorophores by incorporating anintensifier tube with increased sensitivity at short visiblewavelengths. Such ‘high-blue’ tubes are becoming available, and ifapplied could drastically reduce damage and phototoxicity that isassociated with ultraviolet and violet illumination.

In summary, the method, devices and systems of the present inventionadvantageously allow the direct visualization by eye of normallyinvisible, long-wavelength fluorescence through an optical microscopeand also allow visualization of a wide variety of specimens at greatlyreduced illumination levels, regardless of the microscopic techniqueinvolved. Further the results herein with the described examplesindicate the flexibility and potential of the device—by allowing anobserver to see in the dark, it expands the list of observablefluorophores and shields fragile specimens.

Although a preferred embodiments of the method, devices and systems ofthe present invention have been described using specific terms, suchdescription is for illustrative purposes only, and it is to beunderstood that changes and variations may be made without departingfrom the spirit or scope of the following claims.

1. A method for microscopic visualization of a sample comprising: (a)intensifying the light emanating from the sample; and (b) directlyobserving a light image provided by the intensified light.
 2. The methodof claim 1, wherein said light intensifying and observing occurs in realtime.
 3. The method of any one of claims 1 or 2, further comprising thestep of controlling the power output of an illumination source to adesired intensity.
 4. The method of claim 3, wherein the desiredintensity is such that light emanating from the sample is not optimalfor direct visual observation, and wherein said intensifying the lightyields a visible image that is observable to the human eye.
 5. Themethod of any one of claims 1-4, further comprising converting the lightthat is emanating from the sample in non-visible wavelengths of light tolight that is in the visible light spectrum.
 6. The method of claim 5,wherein said observing includes observing the light image provided bythe converted intensified light.
 7. The method of any one of claims 5 or6, wherein the non-visible light is in one of a near-infrared spectralrange or a far-red spectral range.
 8. The method of any one of claims 5or 6, wherein the non-visible light is in the ultraviolet spectralrange.
 9. The method of any one of claims 1-8, further comprisingproviding an visual converter that is configured and arranged so as tointensify the light received at an input end and to provide an image atan output end and wherein said observing includes observing the image atthe output end.
 10. The method of claim 9, wherein the visual converteris located in the optical light path between the sample and an observersuch that the light from the sample is received at the visual converterinput end.
 11. The method of any one of claims 9 or 10, wherein thevisual converter is located so that an input face of a lightintensifying device is one of at or proximal the intermediate imageplane of the microscopic imaging device.
 12. The method of any of any ofclaims 9-11, further providing a plurality of the visual converters, onevisual converter being located in each optical light path so as to allowstereoscopic imaging.
 13. The method of claim 12, wherein the pluralityof the visual converters are arranged so as to allow binocular vision ofthe intensified light.
 14. The method of any one of claims 9-13, whereinthe visual converter is configured and arranged such that light beingreceived at the input end is intensified so as to provide a visibleimage at the output end thereof.
 15. The method of claim 14, furthercomprising the step of controlling the power output of an illuminationsource to a desired intensity.
 16. The method of claim 15, wherein thedesired intensity is such that light emanating from the sample is notoptimal for direct visual observation, and wherein said intensifying thelight yields a visible image that is observable to the human eye. 17.The method of any one of claims 9-16, wherein the visual converter isconfigured and arranged so that light being received at the input endthat is in a non-visible light spectral range is converted to provide alight image that is in the visible light spectral range at the outputend thereof.
 18. The method of claim 17, wherein the non-visible lightis in one of a near-infrared spectral range or a far-red spectral range.19. The method of claim 17, wherein the non-visible light is in aultraviolet spectral range.
 20. An visual converter device comprising:(a) an electro-optical device that is sensitive to light in a wavelengthrange of interest and configured to intensify light in the wavelengthrange of interest focused on its input face to an image that can bedirectly visualized at the output face thereof; and (b) a housing inwhich the electro-optical device is contained, which housing isconfigured and arranged to operably couple the visual converter to amicroscopic imaging device and to minimize external stray light frombeing observed at the output face of the electro-optical device.
 21. Thevisual converter device of claim 20, wherein said electro-optical deviceintensifies the light that is focused on its input face to light that isin the visible light spectral range.
 22. The visual converter device ofclaim 21, wherein the power output of an illumination source iscontrolled to a desired intensity.
 23. The visual converter device ofclaim 22, wherein the desired intensity is such that light emanatingfrom the sample is not optimal for direct visual observation, andwherein said intensifying the light yields a visible image that isobservable to the human eye.
 24. The visual converter device of any ofclaims 20-23, wherein said electro-optical device converts the lightthat is focused on its input face in non-visible light spectral rangesto light that is in the visible light spectral range.
 25. The visualconverter device of claim 24, wherein the non-visible light is in one ofa near-infrared spectral range or a far-red spectral range.
 26. Thevisual converter device of claim 24, wherein the non-visible light is ina ultraviolet spectral range.
 27. A microscopic imaging systemcomprising: (a) a microscopic imaging device; and (b) a visual converterdevice of any one of claims 20-26 operably coupled to the microscopicimaging device, wherein the visual converter device is located in theoptical light path between the sample and an output end of themicroscopic imaging device, such that the light from the sample isreceived at the visual converter input end.
 28. The microscopic imagingsystem of claim 27, wherein the visual converter device is located sothat the input face of the light intensifying device is one of at orproximal to the intermediate image plane of the microscopic imagingdevice.
 29. The microscopic imaging system of any one of claims 27-28,further providing a plurality of the visual converters, one visualconverter being located in each optical light path so as to allowstereoscopic imaging.
 30. The microscopic imaging system of claim 29,wherein the plurality of the visual converters are arranged so as toallow binocular vision of the intensified light.
 31. The microscopicimaging system of any one of claims 29 or 30, wherein saidelectro-optical device intensifies the light that is focused on itsinput face to light that is in the visible light range.
 32. Themicroscopic imaging system of claim 31, wherein the power output of anillumination source is controlled to a desired intensity.
 33. Themicroscopic imaging system of claim 32, wherein the desired intensity issuch that light emanating from the sample is not optimal for directvisual observation, and wherein said intensifying the light yields avisible image that is observable to the human eye.
 34. The microscopicimaging system of any one of claims 28-33, wherein the visual converteris configured and arranged such that light being received at the inputend that is in a non-visible light spectral range is converted so as toprovide a visible image at the output end thereof.
 35. The microscopicimaging system of claim 34, wherein the non-visible light is in one of anear-infrared spectral range or a far-red spectral range.
 36. Themicroscopic imaging system of claim 34, wherein the non-visible light isin an ultraviolet spectral range.